Mitochondrial Ca2+ uptake is central to energy metabolism, cell signaling and dynamics but exploration of the underlying molecular mechanisms and physiology has just started with the discovery of MCU, a pore-forming protein and MICU1, an EF-hand protein described as a critical regulator of the Ca2+ uniporter, which display striking co-evolution and co-expression. Recently, MICU2/3, paralogs of MICU1, MCUb, a dominant negative form of MCU, EMRE, an adaptor for MCU, and MCUR1, another regulator of the uniporter have also been described. Although a permanent and huge driving force supports mitochondrial Ca2+ uptake, there is a strict and complex regulation of the uniporter (mtCU) current by [Ca2+]. Cytoplasmic Ca2+ sensitivity to the MCU is conferred by MICUs via their EF hands. Work by us and others provided evidence that MICU1 is needed to keep the MCU closed at submicromolar cytoplasmic [Ca2+] ([Ca2+]c) and to promote co-operative MCU opening at higher [Ca2+]c. In the past <1.5 year project period, we have established the first mouse model for MICU1 and demonstrated that deletion of MICU1 increases the sensitivity to mitochondrial Ca2+ overload and cell death both in vivo and in vitro. Importantly, human disease associated with MICU1 loss of function has also been described in many patients. Thus, delineation of the mechanism relaying the effect of Ca2+ to the pore, and the functional significance and pharmacological targeting of mtCU are of vast significance. Moreover, because of complementing relevant skills in our group, and our previous success in and powerful toolkit for the study of the uniporter, we are uniquely qualified to move the stick in this area. Our hypotheses are that (1) MICU1 employs 3 distinct interaction sites for binding MCU, EMRE, and MICUs to relay the effect of Ca2+ to control the ion flux across the mtCU, (2) differences in the relative expression level of the mtCU components determine the tissue specific differential Ca2+ sensitivity and pharmacological properties of the mtCU, (3) in the liver and (4) the brain of MICU1-deficient mice, responses to physiological stimulation and responses to various stress conditions can be distinctively affected by impaired gatekeeping and cooperative activation of the mtCU, respectively. Testing of these ideas will provide clues to the fundamental mechanism controlling mtCU, to potential drug targets and to the pathogenesis initiated by perturbation of the mtCU constituents. The studies will utilize both novel human and murine genetic models and imaging methods.